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Our previous studies of protective immunity and pathology against blood stage malaria parasites have shown that not only CD4+ T cells, but also CD8+ T cells and macrophages, are important for host defense against blood stage malaria infection. Furthermore, we found that Plasmodium yoelii 17XNL (PyNL) parasitizes erythroblasts, the red blood cell (RBC) precursor cells, which then express MHC class I molecules. In the present study, we analyzed spleen cytokine production. In CD8+ T cell-depleted mice, IL-10 production in early stage infection was increased over two-fold relative to infected control animals and IL-10+ CD3− cells were increased, whereas IFN-γ production in the late stage of infection was decreased. At day 16 after PyNL infection, CD8+ T cells produced more IFN-γ than CD4+ T cells. We evaluated the involvement of the immunoproteasome in induction of immune CD8+ T cells, and the role of Fas in protection against PyNL both of which are downstream of IFN-γ. In cell transfer experiments, at least the single molecules LMP7, LMP2, and PA28 are not essential for CD8+ T cell induction. The Fas mutant LPR mouse was weaker in resistance to PyNL infection than WT mice, and 20% of the animals died. LPR-derived parasitized erythroid cells exhibited less externalization of phosphatidylserine (PS), and phagocytosis by macrophages was impaired. Furthermore, we tried to identify the cause of death in malaria infection. Blood lactate concentration was increased in the CD8+ T cell-depleted PyNL-infected group at day 19 (around peak parasitemia) to similar levels as day 7 after infection with a lethal strain of Py. When we injected mice with lactate at day 4 and 6 of PyNL infection, all mice died at day 8 despite demonstrating low parasitemia, suggesting that hyperlactatemia is one of the causes of death in CD8+ T cell-depleted PyNL-infected mice. We conclude that CD8+ T cells might control cytokine production to some extent and regulate hyperparasitemia and hyperlactatemia in protection against blood stage malaria parasites. [ABSTRACT FROM AUTHOR]

CD4+CD25+ regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4+CD25+ Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25+ cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25+ cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4+CD25− T cells alone or a combination of CD4+CD25+ and CD4+CD25− T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4+CD25+ Treg cells to CD4+CD25− effector T cells, as demonstrated by the lack of response of CD4+CD25+ T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette–Guérin and M. tuberculosis. Our data show that CD4+CD25+ Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection. [ABSTRACT FROM PUBLISHER]

We investigated the roles of γδ T, NK, and NK1.1+ T-like (NKT) cells in protective immunity against infection with Toxoplasma gondii. γδ T cells, NKT and NK cells, and NK cells in BALB/c mice were depleted by treatment with anti-TCR-γδ monoclonal antibody (mAb), anti-interleukin-2 receptor beta chain (IL-2Rβ) mAb, and anti-asialoGM1 Ab, respectively, and these mice were infected with T. gondii. Treatment of mice with anti-TCR-γδ mAb aggravated toxoplasmosis, while treatment with anti-asialoGM1 Ab had no effects. Treatment with anti-IL-2Rβ mAb enhanced the expression of heat shock protein 65 (HSP65) and gamma interferon (IFN-γ) mRNA, while it inhibited interleukin-4 (IL-4) mRNA expression, ameliorating toxoplasmosis. In addition to NK cells, anti-IL-2Rβ mAb eliminated cells expressing IL-2Rβ and intermediate levels of CD3 (IL-2Rβ+ CD3int). Mice treated with anti-IL-2Rβ mAb decreased the number of DX5+ CD3int cells, which are considered to be equivalent to NK1.1+T cells in NK1.1 allele-negative strains. IL-2Rβ+ CD3int cells isolated from splenic and hepatic lymphoid cells were confirmed to express the TCR-Vα14 transcript. The magnitude of HSP65 induction in macrophages correlated with the protective potential against T. gondii infection after treatment with the antibodies, supporting our previous finding that γδ T cells play an essential role in the induction of HSP65 in host macrophages. Interestingly, NKT cells suppressed the expression of γδ T cell-induced HSP65 and IFN-γ. Furthermore, depletion of IL-2Rβ+ CD3int cells suppressed the IL-4 mRNA expression. These results suggest that NKT cells may be the cells responsible for suppression of protective immunity against T. gondii infection by interfering with the γδ T cell-induced HSP65 expression, possibly through the generation of IL-4. [Copyright &y& Elsevier]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic CD8+ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8+ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8+ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4+ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8+ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi. [Copyright &y& Elsevier]

Abstract: We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8+ T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8+ T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8+ T cells. [Copyright &y& Elsevier]

Abstract: Chagas’ disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8+ T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8+ T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-γ (IFN-γ) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-γ production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas’ disease. [Copyright &y& Elsevier]

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D. [Copyright &y& Elsevier]